The Effect Of Thalidomide On Inflammation And Fibrosis In Mice With Systemic Sclerosis

THE FIRST PART OF THALIDOMIDE ON SYSTEMIC SCLEROSIS MODEL MICE INFLAMMATION AND THE EFFECTS OF PATHOLOGICAL CHANGES IN FIBROSISObjective: To study the effects of thalidomide(Thal)on pathological changes of skin and lung tissue inflammation and fibrosis in mice with systemic sclerosis(SSc).Methods: Eighty female BALB/c mice were randomly divided into a14-day group and a 28-day group(n=40).The SSc mouse model was induced by subcutaneous injection of bleomycin(BLM),respectively,at 10,20,At 30mg/kg/d dose of Thal,the mice in each group were tested on the 14 th and 28 th day of intragastric administration.HE and Masson's staining were used to observe the skin,lung inflammation and fibrosis scores of the back injection site of mice,and the content of hydroxyproline(HYP)was determined.The expression of type I collagen in skin and lung tissues was detected by Rt-PCR and immunohistochemistry.Happening.Result:(1)Both the 14-day group and the 28-day group Thal can reduce the skin and lung tissue of SSc mouse model to different degrees.Inflammation score,14 days skin F value = 23.867,P <0.05,lung tissue F value = 47.811,P <0.05,28 days group skin F value = 34.412,P <0.05,lung tissue F value = 45.953,P<0.05;(2)Thal in the 14-day group and the 28-day group can reduce the skin and lung tissue of the SSc mouse model to different degrees.The degree of fibrosis,in which 14 days group skin F value = 37.698,P <0.05,lung tissue F value =65.500,28 days group skin F value = 72.906,P <0.05,lung tissue F value =35.498,P <0.5;(3)Thal in 14-day group and 28-day group can reduce the skin and lung tissue of SSc mouse model to different degrees.The hydroxyproline content,14 days skin F value = 5.834,lung tissue F value =3.986,P <0.05,P <0.05,28 days group skin F value = 3.631,P = 0.045,lung tissue F value = 6.631,P=0.007;(4)Compared with the 28-day group and the 28-day group,the skin thickness and inflammation score were statistically significant at 10 mg/Kg/d Thal(P=0.039),while the degree of pulmonary fibrosis and inflammation score were only different.There was statistical significance in the treatment of 30mg/Kg/d Thal(P=0.022);(5)Thal can reduce the expression of type I collagen m RNA in lung tissue in a dose-dependent manner in the 28-day group,while Thal 20 and 30(mg/kg/d)can reduce the expression of type I collagen m RNA in the skin(P<0.01).);(6)Thal can reduce the expression of type I collagen in skin and lung tissue in a dose-dependent manner in the 28-day group,but it was more effective at the therapeutic dose of Thal20 and 30(mg/kg/d)(P<0.05)..Conclusion: Thal treatment can effectively reduce the inflammation and fibrosis of skin and lung tissue in SSc mouse model,and can reduce the content of hydroxyproline in skin and lung tissue and inhibit the expression of type I collagen in skin and lung tissue.Therefore,we speculate that Thal may be aneconomical,safe and effective drug for the treatment of patients with SSc.THE SECOND PART OF THALIDOMIDE ON CD4+TH CELLS AND TGF-?1/SMAD3 PATHWAY IN MICE WITH SYSTEMIC SCLEROSISObjective: To observe the effects of different doses of thalidomide(Thal)on the levels of Th1 cells,Th2 cells,Th17 cells,Treg cells and related factors and TGF-?1/Smad3 pathway in systemic sclerosis(SSc)mice.Methods: Eighty female BALB/c mice were randomly divided into a14-day group and a 28-day group(n=40).The SSc mouse model was induced by subcutaneous injection of bleomycin(BLM),respectively,at 10,20,At 30mg/kg/d dose of Thal,the mice in each group were tested on the 14 th and 28 th day of intragastric administration.Flow cytometry was used to detect CD4+IL-4+(Th1)cells,CD4+IFN-?+(Th2)cells,CD4+IL-17+(Th17)cells and CD4+CD25+Foxp3+(Treg)in mouse spleen.The ratio of cells;RT-PCR,immunohistochemistry,enzyme-linked immunosorbent assay and other methods to detect interleukin(IL-)17A,transforming growth factor(TGF)-?1,fork-like transcription factor in peripheral blood,skin and lung tissues(Foxp3),phosphorylated cell signal transduction factor 3(p-Smad3)expression,and then evaluate the mechanism of inflammation and fibrotic lesions in SSc model mice;Western blotting detection of TGF-?1/Smad3 signaling pathway protein expression and phosphoric acid Levels were analyzed and analyzed forassociation with Th17,Treg and its related cytokines.Result:(1)Thal can down-regulate Th17 cells and up-regulate Treg cells in both14-day and 28-day groups(both P<0.05),and Th17 cells can be treated with Thal 30(mg/kg/d)in the 28-day group.The ratio is reduced to the NC group level;(2)Thal in the 14-day and 28-day groups could down-regulate the expression of cytokine IL-17 A m RNA in skin and lung tissue(P<0.05)and up-regulate the expression of transcription factor Foxp3 m RNA(all P<0.05).);(3)Comparison of skin and lung Foxp3 m RNA expression and skin IL-17 A m RNA expression in the 14-day and 28-day groups was statistically significant at 30 mg/Kg/d Thal(P=0.035,P= 0.009,P=0.022),and the expression level of lung IL-17 A m RNA was statistically significant at 20 mg/Kg/d Thal(P=0.005);(4)Thal up-regulated the expression of CD4+IL-4+(Th1)cells and cytokine IFN-? m RNA cells in a dose-dependent manner(both P<0.05),and down-regulated the expression of CD4+IFN-?(Th2)cells(P<0.05);(5)Thal decreased the expression of IL-23 R m RNA and RORgt m RNA in skin and lung tissue in a dose-dependent manner.Correlation analysis showed that IL-23 R m RNA,RORgt m RNA and lung RORgt m RNA levels in skin were positively correlated with Th17(r = 0.9502,0.5664 and 0.7564,P < 0.05);(6)Thal can inhibit the expression of TGF-?1,p-Smad3 protein,TGF-?1m RNA and p-Smad3 m RNA in TGF-?1/Smad3 pathway(all P<0.05);(7)Correlation analysis showed that TGF-?1 m RNA was positively correlated with the expression of IL-17 A m RNA and IL-17 A protein in skin and lung tissues(r values were 0.7280,0.6408,respectively,P<0.01),and Foxp3 m RNA was associated with lung tissue.There was a negative correlation(r=-0.3110,P=0.014);the expression of TGF-?1 protein in skin,lung tissue and serum was positively correlated with the expression of IL-17 A protein(r values were 0.8153,0.7187,respectively,P<0.01).Serum TGF-?1 content was positively correlated with Th17/Treg in spleen(r=0.6366,P=0.0107);Conclusion:(1)Thal can down-regulate the ratio of Th17 cells and the expression of cytokine IL-17 A,up-regulate the ratio of Treg cells and the transcription factor Foxp3,and decrease the ratio of Th17/Treg.(2)Thal treatment can up-regulate the expression of Foxp3 and promote It binds to RORgt,thereby inhibiting the proliferation and function of Th17 cells;(3)Thal treatment can increase Th1 cells and cytokine IFN-? levels,but reduce Th2 cell levels;(4)Thal treatment can inhibit TGF-?1/Smad3 Activation of the signaling pathway to prevent fibrotic lesions.